Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Creation of a Long-Acting Rilpivirine Prodrug Nanoformulation

doi: 10.1016/j.jconrel.2019.09.001

Figure Lengend Snippet: Nanoformulation uptake, retention, and long-term antiretroviral efficacy was assessed in MDMs. Specifically, NM3RPV and NRPV were evaluated for uptake (A,E) and retention (B,F) of intracellular RPV and M3RPV at concentrations of 10 and 30 μM. Data is expressed as mean ± SEM with n = 3 biological replicates (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by Student’s t test). Long-term antiretroviral efficacies of NM3RPV and NRPV were examined after an 8-hour drug treatment with 10 or 30 μM, followed by HIV-1ADA (MOI = 0.1) challenge up to 30 days post treatment. Infection was characterized by HIV-1 reverse transcriptase (RT) activity (C,G) and HIV-1p24 antigen expression (D,H). HIV-1 RT activity was normalized as a percentage of HIV-1 positive control and expressed as mean ± SEM with n = 4 biological replicates. Representative images of MDMs stained for HIV-1p24 antigen expression (brown stain) are shown.

Article Snippet: NM3RPV PK in rhesus macaques Male rhesus macaques (4.4–6.7 kg; PrimeGen) were anesthetized with ketamine (10 mg/kg) and subsequently administered 45 mg/kg RPV-eq. of NM3RPV and a lab-generated CAB prodrug by IM injection (0.5 mL/kg).

Techniques: Infection, Activity Assay, Expressing, Positive Control, Staining

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Creation of a Long-Acting Rilpivirine Prodrug Nanoformulation

doi: 10.1016/j.jconrel.2019.09.001

Figure Lengend Snippet: (A-C) Physicochemical stability of NRPV and (D-F) NM3RPV were evaluated for size (nm), polydispersity (PDI), and zeta potential (mV) at multiple temperatures (room temperature [(RT],), 4 °C and 37 °C) across 100 days. Data is expressed at mean ± SD for n = 3 measurements. Transmission electron microscopy (TEM) provided a morphological assessment of NRPV (G) and NM3RPV (H). (I) Prodrug stability within nanoformulated M3RPV was determined by analyzing the ratio of M3PRV to RPV in NM3RPV over a period of 100 days. Data are expressed at mean ± SEM for n = 3 measurements.

Article Snippet: NM3RPV PK in rhesus macaques Male rhesus macaques (4.4–6.7 kg; PrimeGen) were anesthetized with ketamine (10 mg/kg) and subsequently administered 45 mg/kg RPV-eq. of NM3RPV and a lab-generated CAB prodrug by IM injection (0.5 mL/kg).

Techniques: Transmission Assay, Electron Microscopy

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Creation of a Long-Acting Rilpivirine Prodrug Nanoformulation

doi: 10.1016/j.jconrel.2019.09.001

Figure Lengend Snippet: Male BALB/cJ mice were administered a single intramuscular injection of NM3RPV or NRPV at 45, 75, or 100 RPV-eq./kg (A) RPV concentrations in plasma were determined at days 1 and 7 then weekly for 46 weeks. PA-IC90 and limit of quantitation (LOQ) defined as 12 and 0.5 ng/mL respectively. Tissue biodistribution of RPV and M3RPV was assessed at 56 and 323 days after injection in the (B) spleen, (C) lymph node, (D) liver, (E) gut and (F) kidney. Data is expressed as mean ± SEM where N = 4/5 biological replicates (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 by Student’s t test).

Article Snippet: NM3RPV PK in rhesus macaques Male rhesus macaques (4.4–6.7 kg; PrimeGen) were anesthetized with ketamine (10 mg/kg) and subsequently administered 45 mg/kg RPV-eq. of NM3RPV and a lab-generated CAB prodrug by IM injection (0.5 mL/kg).

Techniques: Injection, Quantitation Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Creation of a Long-Acting Rilpivirine Prodrug Nanoformulation

doi: 10.1016/j.jconrel.2019.09.001

Figure Lengend Snippet: Four rhesus macaques were administered 45 mg/kg RPV-eq. of NM3RPV by a single IM injection. (A) Plasma samples were collected and assayed for RPV and NM3RPV at day 1, 7 and weekly throughout the course of study (316 days). Additionally, rectal, lymph node, and adipose tissue biopsies were collected 204 days after drug administration and assayed for RPV (B) and NM3RPV (C) content. Both plasma and tissue drug concentrations were determined by UPLC-MS/MS. Data are expressed as mean ± SEM for n = 4.

Article Snippet: NM3RPV PK in rhesus macaques Male rhesus macaques (4.4–6.7 kg; PrimeGen) were anesthetized with ketamine (10 mg/kg) and subsequently administered 45 mg/kg RPV-eq. of NM3RPV and a lab-generated CAB prodrug by IM injection (0.5 mL/kg).

Techniques: Injection, Tandem Mass Spectroscopy

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: In vitro expression analysis of hepatitis B surface antigen mRNA vaccines. LNP-encapsulated L mRNA, M mRNA, and S mRNA were transfected into 293T cells in different combinations, and the dosage of each single mRNA was 5 μg for each treatment. ( a ) The proportion of cells expressing pre-S2 antigen or S antigen on the surface was detected by FCM at 48 h and 96 h after transfection, respectively. The results of FCM were showed using density maps. ( b ) WB analysis of the expression or secretion of the L protein (42 kDa), M protein (31 kDa), and S protein (27 kDa) in the cell membrane or supernatants at 48 h after transfection.

Article Snippet: Subsequently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of IgG to different hepatitis B antigens as follows: a. Pre-S peptide and recombinant HBsAg (671-01, Shanghai PrimeGene Bio-tech Co., Shanghai, China) were diluted to 1 μg/mL in NaHCO 3 buffer (pH 9.6), and the LMS VLP were diluted to 10 μg/mL.

Techniques: In Vitro, Expressing, Vaccines, Transfection, Membrane

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Humoral immune response analysis of HBV Tg mice immunized with the hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for the immunization of BALB/c HBV Tg mice with different combinations of mRNAs encoding hepatitis B surface antigens or with control vaccines. ( b ) CLIA detection of serum HBsAb levels in HBV Tg mice at weeks 0–32 of the immunization experiment. ( c ) Analysis of changes in the serum HBsAb concentration in mice whose HBsAb concentration was consistently less than 2000 IU/L and greater than 0 IU/L. ( d ) Correlation analysis between the serum levels of HBsAb and reduced HBsAg levels. ( e ) Indirect ELISA analysis of the binding ability of induced serum IgG to recombinant HBsAg, the pre-S peptide, or the LMS VLP after immunization with different vaccines. ( f ) Variable slope (four parameters) analysis via nonlinear regression (curve fit) was performed for the pre-S antigen-specific indirect ELISA. Representative results are presented as the means ± standard deviations (SDs).

Article Snippet: Subsequently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of IgG to different hepatitis B antigens as follows: a. Pre-S peptide and recombinant HBsAg (671-01, Shanghai PrimeGene Bio-tech Co., Shanghai, China) were diluted to 1 μg/mL in NaHCO 3 buffer (pH 9.6), and the LMS VLP were diluted to 10 μg/mL.

Techniques: Vaccines, Control, Concentration Assay, Indirect ELISA, Binding Assay, Recombinant

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Serological and virological response analysis of pAAV HBV1.2 mice immunized with hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for immunization of pAAV HBV1.2 mice with LMS mRNA vaccines, S mRNA, or GFP mRNA. Changes in serum ( b ) HBsAg, ( c ) HBsAb, and ( d ) HBeAg from 0 to 5 weeks after immunization of pAAV HBV-1.2 mice with different vaccines. ( e ) Q–PCR detection of serum HBV DNA levels in immunized mice at week 0 and week 5. Representative results are presented as the means ± SDs.

Article Snippet: Subsequently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of IgG to different hepatitis B antigens as follows: a. Pre-S peptide and recombinant HBsAg (671-01, Shanghai PrimeGene Bio-tech Co., Shanghai, China) were diluted to 1 μg/mL in NaHCO 3 buffer (pH 9.6), and the LMS VLP were diluted to 10 μg/mL.

Techniques: Vaccines

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Serological and virological response analysis of rAAV8 HBV1.3 mice immunized with hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for immunization of rAAV8 HBV1.3 (1 × 10 10 vg/mouse) mice with hepatitis B surface antigen mRNA vaccines in different combinations, S mRNA, or GFP mRNA. Changes in serum ( b ) HBsAg, ( c ) HBsAb, and ( d ) HBeAg from 0 to 9 weeks of immunization with different vaccines. ( e ) Q–PCR detection of serum HBV DNA levels in immunized mice at week 0 and week 9. Representative results are presented as the means ± SDs.

Article Snippet: Subsequently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of IgG to different hepatitis B antigens as follows: a. Pre-S peptide and recombinant HBsAg (671-01, Shanghai PrimeGene Bio-tech Co., Shanghai, China) were diluted to 1 μg/mL in NaHCO 3 buffer (pH 9.6), and the LMS VLP were diluted to 10 μg/mL.

Techniques: Vaccines

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: The analysis of antigen-dependent T-cell responses activated by Hepatitis B surface antigen mRNA vaccines. ( a ) BALB/3T3 cells were transiently transfected with pD2531.L, pD2531.M, and pD2531.S plasmids (1:1:1 mass ratio) via Lipofectamine 3000, and FCM was performed to detect pre-S2 antigen and S antigen expression on the cell surface. ( b ) After immunization of BALB/c mice with different hepatitis B surface antigen mRNA vaccines or PBS, splenic CD4 + T cells were isolated and stimulated with BALB/3T3 cells transiently expressing the LMS antigen. Meanwhile, antigen-incubated T cells from PBS-immunized mice were used as negative control and CD3/CD28 bead-stimulated T cells from PBS-immunized mice were used as positive control. FCM was used to measure the level of IFN-γ in CD4 + T cells after the addition of brefeldin A.

Article Snippet: Subsequently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of IgG to different hepatitis B antigens as follows: a. Pre-S peptide and recombinant HBsAg (671-01, Shanghai PrimeGene Bio-tech Co., Shanghai, China) were diluted to 1 μg/mL in NaHCO 3 buffer (pH 9.6), and the LMS VLP were diluted to 10 μg/mL.

Techniques: Vaccines, Transfection, Expressing, Isolation, Incubation, Negative Control, Positive Control

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Schematic diagram of the mechanism by which the LMS mRNA therapeutic vaccine disrupts HBsAg-mediated immune tolerance and reactivates immune responses against HBV. The numbers 1–6 marked on the membrane structure of L protein represent intracellular structure of S antigen, T cell recognition epitopes of pre-S1 antigen, hepatocyte surface receptor NTCP binding site for pre-S1 antigen, S antigen (highly variable region) with a-antigen determinant, lipid membrane, hepatocyte binding region for pre-S2 antigen, membrane attachment region of HBV-infected hepatocytes, respectively.

Article Snippet: Subsequently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of IgG to different hepatitis B antigens as follows: a. Pre-S peptide and recombinant HBsAg (671-01, Shanghai PrimeGene Bio-tech Co., Shanghai, China) were diluted to 1 μg/mL in NaHCO 3 buffer (pH 9.6), and the LMS VLP were diluted to 10 μg/mL.

Techniques: Membrane, Binding Assay, Infection